(i) Field of the Invention
This invention relates to a process for the preparation of oligofructans and to the mixture of such oligofructans (i.e. the fructose syrup) so produced.
(ii) Description of the Prior Art
Starch conversion syrups are conventionally produced by the hydrolysis of starch. These starch conversion syrups, e.g. corn syrup, consist primarily of D-glucose, maltose, a small amount of oligoglucans and dextrins. The amounts of these primary constituents, of course, vary from one syrup to another depending upon a number of different factors. The isomer of glucose known as fructose may be formed by the interconversion, i.e. isomerization, of glucose.
It is also known that when a syrup containing sucrose is hydrolyzed, equal parts of fructose and glucose are formed and the product is known as "invert syrup". Sucrose is deliberately converted to invert sugar in the manufacture of certain non-crystallizing syrups, e.g. golden syrup and treacle.
Fructose is also widely distrubuted in nature. Fructose occurs in both furanose and pyranose forms. An aqueous solution at 20.degree. contains about 20% of the furanose form: ##STR1##
It is well known that crystalline fructose is 1.8 times sweeter than sucrose (See, e.g. Shallenberger et al, SUGAR CHEMISTRY, page 116 (1983), the AVI Publishing Company, Inc.) Thus, fructose is fast becoming one of the most popular candidates for sweetening foods and beverages, since its greater sweetening power makes possible a significance reduction in the caloric intake of the food or beverage consumer. Moreover, fructose is preferred to sucrose (cane sugar) in the food industry because it crystallizes less rapidly than sucrose (thus giving a smoother texture). Since its metabolism in humans does not depend on insulin, diabetics can use fructose as a sweetener.
In recent years, a number of synthetic sweeteners have come under close scrutiny as a result of experiments indicating carcinogenic activity in experimental animals; hence, the purely "natural" route to lower caloric intake offered by fructose sweetening has acquired even greater significance.
While sucrose and glucose are produced on a large scale for use in high energy foodstuffs and a great number of technical products, the commercial use of fructose has been very limited up to date, since it has been available mostly as low fructose-content sugars. In many aspects of the industrial practice of manufacturing pharmaceuticals, foods, beverages, dietary supplements, and the like, those various relatively low fructose-content sugars (typically about 42 to about 55% fructose) are not preferred. One somewhat more preferred form would be "high fructose syrup", i.e. a relatively concentrated aqueous solution of substantially pure fructose or fructose mixed with minor amounts of other carbohydrates, which can, if desired, be crystallized directly to obtain substantially pure crystalline fructose.
Another commercial potential of fructose is related to the fact that fructose may be readily converted to mannitol. In the present method of manufacturing mannitol from invert syrup, for every 1 part molar of mannitol produced 3 parts molar of sorbitol are also obtained as a byproduct. On the other hand, a pure fructose syrup yields only 1 part molar of sorbitol for every part molar of mannitol.
In the prior art attempts have been made to utilize the interconversion reaction referred to above to produce syrups sweeter than conventional starch conversion syrups. Various processes for achieving this interconversion have been described in the literature.
The isomerization of D-glucose, D-mannose, and D-fructose by the action of aqueous alkali has long been known. This isomerization reaction is conventionally referred to as the "Lobry de Bruyn-van Ekenstein Conversion" after its discoverers (Rec. trav. chim. Pays-Bas 14.203/1895 and 15.92/1896). It has been found that D-glucose can be isomerized using various catalysts, e.g. sodium hydroxide, sodium carbonate, calcium hydroxide, alkaline earth carbonates, alkaline ion exchangers, ammonia, or pyridine. However, the amounts of D-fructose thereby formed amounted to a maximum of about 10-about 30%, the yields of actually isolated fructose being still lower as the isolation of the fructose from the reaction mixture could be performed only with great difficulty and with considerable losses.
It has been proposed to increase the yields of D-fructose obtained by the alkaline isomerization of D-glucose by working in the presence of borates (Mendicino, J. Am. Chem. Soc. 82.4975/1960). However, this process cannot be used industrially for the production of D-fructose since its isolation from the reaction mixture is practically impossible.
There are many patents in existence directed to attempts to improve the process discovered by Lobry de Bruyn and W. A. van Ekenstein so that such process could be exploited industrially for the manufacture of fructose.
For example, Canadian Pat. No. 488,178 issued Nov. 18, 1952 to Corn Products Refining Company is related to the production of a levulose-containing syrup by the interconversion of dextrose, under the influence of an alkaline catalyst under controlled conditions. In an attempt to overcome the presence of various objectionable substances present in a syrup so produced, namely mannose, a group of non-fermentable sugars known as "glutose", saccharinic acids, colloidal materials, various metallic salts, hydroxymethylglyoxan and methylglyoxal, which imparted an undesirable color and unpleasant taste to the syrup, the patentee added an additional step. The patented process involved the step, with or without removal of the alkaline catalyst, of treating the resultant liquor with a hydrogen base exchanger and acid absorbent resins to remove therefrom various objectionable substances, notably methylglyoxal and hydroxymethylglyoxal.
Canadian Pat. No. 694,539 issued Sept. 15, 1964 to C. F. Boehringer & Soehne, taught that D-fructose could be readily prepared in excellent yields if the isomerization of D-glucose by the action of aqueous alkali is carried out using an alkali metal aluminate, e.g. sodium aluminate or potassium aluminate. When the isomerization was completed, the aluminum was precipitated in the form of its hydroxide and was removed. The D-fructose could then be isolated as calcium fructosates. The D-fructose could be liberated from the fructosate with carbonic acid, followed by the removal of the water by distillation, and recrystallation out of methanol.
Canadian Pat. No. 868,346 issued Apr. 13, 1971 to Laevosan provided a process for the production of fructose and glucose from sucrose. According to that patented process, sucrose was first inverted under mild conditions and the pure invert sugar solution was evaporated under mild conditions. The invert sugar concentrate obtained was treated, preferably after separation from crystallized glucose, with a lower alcohol, e.g. methanol, and the two sugars were crystallized alternately after inoculation with substantially complete separation of the crystals from each other.
Canadian Pat. No. 898,246 issued Apr. 18, 1972 to Ryoki Tatuki, et al, provided a method for separating fructose advantageously from a sugar solution mixed along with glucose, e.g. fructose from the invert sugar solution of sugar, from the isomerized sugar solution obtained by isomerizing glucose at high yield. The patented method involved adding calcium chloride to that sugar solution in the neutral or acidic region, to dissolve calcium chloride therein, condensing the thus obtained mixture solution, forming fructose calcium chloride double salt by slowly cooling that solution while slowly stirring the same, and separating fructose from the double salt. Glucose contained in the thus obtained residual liquid could be recovered by subjecting the raw material sugar solution as the condensing liquor to electrodialysis.
Canadian Pat. No. 1,146,102 issued May 10, 1983 to American Crystal Sugar Company provided a process for obtaining fructose from a fructofuranoside-containing starting material (e.g. a material containing saccharides of the fructofuranoside type), which involved the following steps: (a) hydrolyzing the saccharide to provide a mixture of glucose and fructose; (b) adding a basic calcium compound to precipitate a mixture of calcium-sugar complexes which will comprise primarily the calcium-fructose complex; (c) treating the calcium-fructose complex thereby obtained with phosphoric acid in an aqueous reaction medium under temperature conditions sufficiently cool to provide a high quality product in high yield; and (d) recovering fructose of relatively high purity is from this reaction medium.
Another technique for the preparation of fructose involved chemical precipitation of fructose to separate the products of the inversion reaction. This technique took advantage of the fact that fructsate complexes were less soluble in water than, for example, the corresponding glucosates.
At first glance, such chemical precipitation techniques (whereby alkaline earth metal cations form complexes with the sugars in the sugar mixture) would appear to be very promising. Hydrolysis of the sucrose molecule provided an equimolar mixture of glucose and fructose. Significant progress in the utilization of the chemical precipitation technique for separating fructose from glucose was, however, hindered by the stability of the alkaline earth metal fructose complex under cold conditions. Various acids were found to break up this complex and release the fructose, the most common of these being carbonic acid. Carbon dioxide as carbonic acid caused a precipitation of calcium carbonate and released fructose to the aqueous medium. The calcium carbonate precipitate could then be removed by filtration.
The conventional process is thus based on the precipitation of calcium fructosate by the addition of calcium hydroxide to the invert sugar solution. The highly insoluble frutosate is separated, then split with acids, mostly carbonic acid, and after concentration of the diluted frutose solution obtained, crystallized from methanol.
The results of such carbonic acid precipitation technique have apparently not met modern industry standards for the production of relatively pure fructose for a number of reasons. For example, even at low temperature, a considerable amount of color bodies tended to form prior to or during or even subsequent to the liberation of the fructose from the fructosate complex, due to the destruction of the fructose molecule. In many large scale uses of fructose, a clear solution or a pure white powder or crystal was desirable or even essential for consumer acceptance or for satisfying industry-imposed quality control standards. Consequently, these color bodies must be removed, or their formation avoided.
Patents have issued directed to the separation of the products of sucrose inversion by the formation of a sparingly-soluble salt with an alkaline earth metal hydroxide, e.g. calcium, strontium or barium hydroxide, separation of the precipitate and regeneration thereof to give a sugar solution. The precipitation process with alkaline earth metal hydroxides required a large amount of the hydroxides and, subsequently, a large amount of precipitating agent, for example, carbon dioxide, sulphuric acid or phosphoric acid for the metals. Even when, for example, the precipitated alkaline earth metal carbonates could be regenerated by calcination, a considerable expenditure on auxiliary chemicals and treatment costs was necessary. Furthermore, the sugar solutions obtained were adulterated with impurities, especially ions of the alkaline earth metal used, and must be freed from these, for example, by ion exchangers. However, the ion exchangers, in the acidic form, had a hydrolytic action upon sucrose and, in the alkaline form, caused discolouration.
Other patents teach the use of the easy oxidizability of glucose to gluconic acid by means of bromine or iodine to separate it from the fructose. The gluconic acid is separated off as a sodium or calcium salt which is highly insoluble in methanol, or removed by anion exchangers and the fructose is obtained as above from the remaining solution. Only the electrolytic variant of this principal is technically important due to the high price of the halogens and the high salt loading (see Sugar Research) Foundation, U.S. patent specification No. 2,567,060).
Other patents have issued directed to the separation of the products of sucrose inversion by a suitable pretreatment, for example, with sulphuric acid in an organic solvent, e.g. methanol or ethanol, after which a part of the sucrose can be crystallized from the organic solvent. For example, fructose may be separated from an alcohol solution in the form of its calcium chloride double salt. It was necessary to use a large amount of alcohol and to keep the concentration of alcohol constant in view of the separation of fructose. Moreover, it was necessary to provide equipment for recovering the used alcohol.
In such conventional method in which alcohol solution was used, it was also necessary, in order to separate fructose from the fructose calcium chloride double salt, to add a precipitant for producing the insoluble salt of calcium, e.g. carbonate, sulfate, or oxalate. Then, it was necessary to desalt. Since the amount of calcium chloride contained therein was relatively large, this was not economically viable. In addition, in such method, the loss of fructose was large. It was also known in principle to affect a separation of fructose and glucose by bringing their aqueous solution into contact with an ion exchange resin. One such ion exchange resin was a calcium sulphonated polystyrene cation exchange resin. Fructose was preferentially absorbed by the resin and glucose preferentially remained in the surrounding aqueous liquid. The fructose was subsequently washed out of the resin after displacing the surrounding glucose-enriched solution.
Sucrose could also be hydrolyzed with cationic exchange resins in the H-form, but in such case a complete hydrolysis was only possible with very long residence times of the sucrose in the exchanger. It was further known that invert sugar solutions on exchangers in the pure H-form suffered undesirable discoloration at elevated temperatures and comparatively long residence times. The same thing happened with glucose and fructose solutions which had been produced by inversion with mineral acids and subsequently passed over a basic exchanger for the purpose of removing acid ions.
In another use of ion exchange resins, prior to isomerization glucose-containing liquors were refined by conventional means, e.g., by treating the liquors with carbon and ion exchange materials.
Among the patents directed to such procedures are:
German Pat. No. 2,160,919 to Takasaki taught process for the separation of a mixture of carbohydrates by treating the mixture with an anion exchanger in the sulfite or bisulfite form.
Canadian Pat. No. 525,394 issued May 24, 1966 to American Cyanamid Company provided a procedure for the clarification of aqueous solutions containing a sugar. The invention involved passing an aqueous solution of a sugar through a bed of an anion active resin, and thereafter heating the juice to precipitate insoluble calcium and magnesium salts together with colloidal materials, clarifying or filtering the juice optionally crystallizing sugar therefrom.
Canadian Pat. No. 756,575 issued Apr. 11, 1967 to the Colonial Sugar Refining Company Limited provided a process for the separation of fructose and glucose from syrups containing them, involving a complicated series of recycling steps based upon a first step of sequentially admitting predetermined volumes of the syrup and water to a column charged with a water-immersed bed of an alkaline earth metal salt of a cross-linked cation exchange resin, then separating that effluent into six fractions, then sequentially admitting only two of such fractions along with water and the syrup being separated.
Canadian Pat. No. 1,156,951 issued Nov. 15, 1983 to Nabisco Inc. provided a process for isomerizing glucose in a glucose-containing liquor to fructose, by first treating a glucose-containing liquor with an ion exchange material and then contacting the treated liquor with immobilized glucose isomerase to convert a portion of the glucose to fructose. The glucose-containing liquor was treated with ion exchange material in the bisulfite/sulfite form and the treated liquor was contacted with immobilized glucose isomerase under glucose isomerizing conditions to convert a portion of the glucose in the liquor to fructose. The glucose-containing liquor preferably was one which had been ion-exchage refined.
Canadian Pat. No. 771,127 issued Nov. 7, 1967 to C. F. Boehringer Soehne provided a process for obtaining pure glucose and fructose from sucrose or from sucrose-containing invert sugars by passing an aqueous solution of sucrose or sucrose-containing invert sugar over an ion exchanger still containing between about 1 to about 30% of free acid groups.
Canadian Pat. No. 813,297 issued May 20, 1969 to Boehringer Mannheim GmbH provided a process for the production of invert sugar solutions from molasses, including the steps of first subjecting molasses to acidic hydrolysis at a pH of about 1-about 4, neutralizing the product with an aqueous basic solution or with a weak basic anion exchanger and subsequently separating the products chromatographically on a cation exchange resin solumn in the salt form.
Canadian Pat. No. 877,950 issued Aug. 10, 1971 to Corn Products Refining Company provided sweet syrup products by deanionization of the dextrose-bearing starting material prior to interconversion. Such deanionization removed the mineral anions (e.g. Cl.sup.-, SO.sub.4.sup.--), normally present in the dextrose-bearing starting material and replaced them with OH.sup.- ions. The deanionization also adjusted the pH of the material to the range said to be required for effective interconversion, i.e. about 8.50 to about 10. The deanionization was achieved by the use of a strongly basic anion exchanger or, by the use of an electrodialysis unit.
Canadian Pat. No. 918,150 issued Jan. 2, 1973 to Boehringer Mannheim GmbH provided a chromatographic process for the separation of carbohydrate solution containing glucose and fructose. In the patented process, a carbohydrate solution containing glucose and fructose was allowed to flow through a chromatographic column containing a separatory material adapted to fractionate the sugar into a glucose and a fructose fraction, by an eluting agent.
Canadian Pat. No. 963,899 issued Mar. 4, 1975 to Standard Brands Inc. provided a refined fructose-containing solution produced by an enzymatic process. In the patented process, an enzymatically-produced fructose-containing solution which contained color and color-forming bodies was treated with carbon to remove substantially the major portion of the color and color-forming bodies therefrom. The solution was maintained at an acidic pH, and was treated with a strong acid cation exchange resin in the hydrogen form and a weak base anion exchange resin in the free base form to remove substantially all the remaining color and color-forming bodies.
U.S. Pat. No. 2,746,889 issued May 22, 1956 to A. E. Staley Manufacturing Company described an interconversion process wherein the reaction was effected in the presence of a high basic ion exchange resin and an inert gas in order to deal with the problems of undesirable byproduct formation in the interconversion process. This patent, however, presented the problem of providing and maintaining an inert atmosphere. Moreover, it was characterized by an undesirable loss of dextrose by conversion to acid.
Dow Chemical Company, U.S. patent specification Nos. 3,044,904, 3,044,905 and 3,044,906, attempted to bring about the separation of glucose and fructose by column chromatography of an aqueous invert sugar solution over alkaline earth salts from cation exchangers, where the fructose is retained as opposed to the glucose. Both sugars could be obtained individually in successive eluate fractions. For example, U.S. Pat. No. 3,044,904 taught that glucose and fructose could be separated from aqueous solutions with a cation exchanger of the cross-linked sulphonated polystyrene type charged with calcium ions. Such process only gave good results when an approximately 50% sugar solution was allowed to run through a sufficiently long exchanger column at about 60.degree. . To be operative, the starting material would have to be free from impurities, for example, inorganic salts. Therefore in the case of the known hydrolysis of sucrose with mineral acids, either the acid ions must be removed by an anion exchanger or the hydrolysis must be carried out in known manner with a cationic exchange resin in the H-form.
U.S. Pat. No. 3,285,776 issued Nov. 15, 1966 to Anheuser-Busch, Incorporated described an interconversion process employing alkali in the reaction, wherein the pH was continuously maintained within prescribed limits during the interconversion.
Other procedures proposed involved the enzymatic inversion to fructose. Since the issuance of the pioneer patent in this field, U.S. Pat. No. 2,950,228, granted to Richard O. Marshall on Aug. 23, 1960, there has been a great amount of activity in connection with enzymatic isomerization. Several different microbial sources of glucose isomerase enzyme preparations have been identified.
The conversion of glucose into syrups which contain glucose and fructose can be achieved by exploiting enzymes extracted from a number of micro-organism of the genera Pseudomonas, Lactobacillus, Escherichica, Aerobacter, Bacillus and others, e.g. Aerobacter cloacas, Bacillus megaterium, Acetobacter suboxydans, Acetobacter malanogenus, Acetobacter roseus, Acetobacter oxydans, Bacillus fructosus and Lactobacillus formenti. For the enzymatic isomerization of glucose into fructose, glucose isomerase (D-xylose-ketol-isomerase, 5.3.1.5) may be used to isomerize a solution of glucose, e.g., as corn syrup, under reaction conditions controlled in such a way that a fraction of glucose was converted into fructose, the amount of glucose which is converted into fructose being a function of an equilibrium constant which, at 60.degree. C., is 1.
Thus, as taught by the prior art, starch may be first liquefied by an acid treatment and then saccharification be effected by enzymatic means; or both liquefaction and saccharification may be effected by enzymatic means.
The stability or effective life of immobilized glucose isomerase is probably influenced to the greatest extent by the quality of the substrate. The quality of glucose-containing liquors produced in the corn wet milling industry may be highly variable. Generally, these liquors are refined by conventional methods prior to isomerization. To attempt to avoid this problem, investigations have been carried out relating to the use of enzyme preparations in insoluble form, particularly with respect to the development of continuous isomerization processes.
The use of microbial and fungal enzymes adsorbed onto or bonded to inert carriers to provide immobilized biological catalysts is now prevalent. In general, immobilized enzymes provided a number of significant advantages over soluble or cell-bound enzymes particularly in commercial systems for carrying out continuous conversion processes. Pretreatments were also suggested to remove products which might inactivate enzymes. It has been found, however, that although such treatments provided some prolongation of the effective life of immobilized glucose isomerase, the stability of the enzyme is not as great as is desirable in continuous processes for isomerizing glucose to fructose.
Practicing such processes resulted in an enzymatically-produced fructose-containing solution which had minimal quantities of unwanted byproducts, color bodies and color-forming bodies.
There have been many patents directed to such enzymatic procedures. For example, U.S. Pat. Re. No. 28,885 to Cotter et al. provided an enzymatic method for isomerizing glucose syrups utilizing soluble glucose isomerase or cellular material containing this enzyme. Incorporation of a source of SO.sub.2 into glucose-containing liquors during isomerization e.g. by soluble salts of sulfurous acids or by passing the liquor through ion exchange was taught to reduce denaturation of the glucose isomerase and to inhibit undesirable color formation in the finished product.
British Pat. No. 1,103,394 and Japanese Pat. No. 7428 (1966) to Takasaki et al. disclose that microorganisms classified as belonging to the Streptomyces genus, such as Streptomyces flavorirons, Streptomyces achromogenes, Streptomyces echinatus and Streptomyces albus albus produce glucose isomerase.
In Die Starke, 26 Jahrg., 1976/Nr. 10, pp. 350-356, Oestergaard et al. recommended that glucose-containing substrates be filtered and treated with carbon and ion-exchange materials prior to carrying out continuous isomerizations with glucose isomerase to remove impurities which may adversely affect the activity of the enzyme. They further disclosed that possibly harmful enzyme contaminants in the syrup, which apparently were formed during isomerization, may be protected against by utilizing a particular arrangement of a plurality of columns containing the immobilized glucose isomerase.
Canadian Pat. No. 986,866 issued Apr. 6, 1976 to CPC International Inc. provided a procedure for the isomerization of starch hydrolysates that contain glucose, to produce levulose-bearing products, by enzymatic isomerization, by the action of xylose isomerase (E.C. 5.3.1.5) enzyme preparation under non-oxidizing conditions. The glucose isomerase enzyme preparation was produced from a Streptomyces microorganism, for example, S. olivochromogenes.
Canadian Pat. No. 947,217 issued May 14, 1974 to Ken Hayashibara provided processes for the production of oligosaccharide mixtures having fructose molecules on their reducing ends (oligosyl fructose) by subjecting mixtures of starch, sucrose or fructose to the action of specific alpha-amylases thereby attaining simultaneous hydrolysis of starch and transfer of the formed oligosaccharides into sucrose or fructose.
Canadian Pat. No. 1,106,225 issued Aug. 4, 1981 to Snamprogetti provided a method for the production of fructose and syrups containing fructose and glucose by using an isomerizing enzyme obtained from microorganism of the Streptomyces genus. The patented involved contacting a solution of glucose with a micro-organism selected from the group consisting of Streptomyces sp. genus NRRL 11.120 and NRRL 11.121 or with the enzyme originated thereby.
Canadian Pat. No. 1,117,047 issued Jan. 26, 1982 to CPC International Inc. provided a procedure for the enzymatic transfructosylation of sucrose by way of a fructose polymer-containing substrate. By the patented process a primary substrate, e.g., sucrose, was subjected to the action of a specified fructosyl transferase enzyme preparation.
As noted above, as a result of the above prior art discussion it is clear that fructose, a sugar of great sweetness and general utility, has hitherto been made only at high cost. It could also be made in small quantities by acid hydrolysis of plant polyfrucosans (e.g. inulin, found in Jerusalem artichokes, dahlias and certain other plants). It is known that inulin of the following structure, yields D-fructose and D-glucose by adding absolute alcohol to the syrup obtained from acid hydrolysis: ##STR2##
Some researches claim that a variety of physiological benefits can be obtained by including fructose in the diet. Thus, recent dietary experiments in Japan have shown two favourable effects of the fructooligosaccharides (GF.sub.2-4) in humans. Five week administration of a glucose syrup containing the oligosaccharides to atherosclerosis patients resulted in a significant decline in blood cholesterol and blood pressure. Two week administration of the syrup to geriatric patients increased the population of a gut bacterium, Bifidobacterium, to the levels found in healthy younger humans (the decline in population of the bacterium has been associated with aging). Bifidobacterium grows well on these oligosaccharides (which are not digestible by humans) and produces lactic and acetic acids to lower the pH of the intestine. An increase in the intestinal pH has been associated with aging and is responsible for formation of nitrosamines which are potential carcinogens.
Because of these desirable physiological effects of the oligofructans, Meiji Seika Co. is manufacturing a "glucose" syrup containing these oligosaccharides produced by treating cane sugar (sucrose) in an immobilized fungal fructosyltransferase column. Its average composition is:
______________________________________ Monosaccharide, largely glucose (G) about 37% Sucrose (GF) about 11% GF.sub.2 about 24% GF.sub.3 about 23% GF.sub.4 about 5% ______________________________________
As glucose is less sweet than sucrose and GF.sub.2-4 are much less sweet, the sweetness of such product is only about 60% of that of sucrose.
Despite the apparent recognition that an acceptable process for producing sweet syrups by interconversion would necessarily provide for the elimination or careful control of undesirable by-product and color formation, it appears that no practical solution has heretofore been developed.
Although the prior art methods have proven to be beneficial to a degree, continuous isomerization processes utilizing immobilized glucose isomerase have not hitherto been as efficient as desired due to the fact that the enzyme becomes inactivated after a relatively short period of use. Although the enzymatically-produced fructose-containing solutions produced by the methods described herein were relatively pure, it was nevertheless still necessary to refine the same in order to remove color, color-forming bodies and salts therefrom.